Method of orally administering a fecal microbiota transplant composition

ABSTRACT

Disclosures herein relate to a fecal microbiota transplant (FMT) composition for oral delivery, the FMT composition comprising an FMT including desired fecal microbes, wherein the FMT has an odor as evaluated with a confidence of 95% to be odorless for more than 50% of the population using a triangular test, wherein the FMT has a flavor as evaluated with a confidence of 80% to be flavorless for more than 50% of the population using the triangular test.

CLAIM TO PRIORITY

This application claims the benefit of priority to U.S. ProvisionalApplication 63/370,034, filed Aug. 1, 2022 (NOVL-0001-P01). Theforegoing application is incorporated herein by reference in itsentirety for all purposes.

BACKGROUND Field

The present disclosure relates to fecal microbiota transplantcompositions and methods for treating patients.

Description of the Related Art

The presence of normal, healthy, intestinal microbiota may offerprotection against various illnesses, such as C. difficile infections.Conversely, dysbiotic gut microbiomes may be linked to certain disordersand pathologies, such as autism spectrum disorder and recurrent C.difficile infections. Fecal Microbiota Transplant (FMT) provides apathway to the restoration of a healthy gut. Encapsulated FMTcompositions have been developed, however, there are challengesassociated with providing FMT therapy to patients who are unable toswallow capsules and/or who do not want or cannot access the treatmentby other established treatment methods (e.g., capsule, enema, endoscopy,colonoscopy, NJ-tube, etc.). Thus, there remains a need for an orallydosed FMT composition that is virtually colorless, odorless, andtasteless.

Efforts have been made to provide odorless and tasteless FMTcompositions for oral delivery, but none have been successful atadequately suppressing odor and taste of fecal matter components toallow adequate compliance for administration protocols that requiresignificant loading doses. Specifically, it is known that variousfiltration techniques are used as one means of attempting to isolatefecal matter microbes, but those techniques have not successfullyachieved compliance with oral loading dose administration.

Another means involves culturing and growing the microbes in a separateand clean environment, but with given technologies and the amount andvariety of microbes needed for a successful loading dose andengraftment, this is extremely cost prohibitive and, to date has notbeen proven to be as successful. Of course, it would be ideal to have apristine source of desired fecal microbes that have never encounteredactual fecal matter for the purpose of oral delivery, but this is asolution that is not remotely close to having a reasonable cost basis orproven successful for use in therapeutic protocols that require dosingwith billions or trillions of microbes.

To the best of the inventor's knowledge, the state of the art asreflected in review articles relating to FMT at the time of filing thispatent application showed that oral delivery of FMT was only practicallyachievable in encapsulated form. In other words, at the time of filingthis patent application, the state of the art for oral delivery of FMTrequires encapsulation, such as to provide a physical barrier betweenthe FMT composition itself and the subject's tongue and stomach, toprovide protection against thermal stress, to provide a moisture barrierduring storage, to provide protection for labile components, and thelike. This provides meaningful limitations to compliance, because manyindividuals are unable to swallow capsules without significant effort.

Other approaches to administering foul-tasting and/or foul-smellingtherapeutics can be applicable to oral delivery of FMT, includingmasking flavor and odor (e.g., co-delivery with chocolate milk). Thoseapproaches make more sense in circumstances where the underlyingtherapeutic is itself the source of the foul taste and/or smell. In thiscase, however, it is believed that the desirable gut microbes themselvesare not responsible for the foul taste and/or smell associated withtheir potential oral delivery, so the entities that are responsible forsaid taste and/or smell might be capable of being diminished whileretaining adequate quantity and quality of desired gut microbes.Applicant is aware that such flavor- and odor-masking agents have beenused with compositions that purport to be useful for the delivery of gutmicrobes. Applicant submits that the majority of practitioners in thefield would presume that an assertion that a composition is odorlessand/or flavorless does not have merit if the composition is onlyadministered with flavor- and/or odor-masking agents and there are noother test results showing lack of odor or flavor.

A need exists for orally deliverable FMT compositions that facilitateadministrative protocols delivering significant quantities of desirablegut microbes.

SUMMARY

Provided herein are FMT compositions used for oral dosing that arevirtually colorless, tasteless, and odorless, and related methods formanufacturing FMT compositions are disclosed. The disclosed compositionsprovide a dosing mechanism for patients who are unable to swallowcapsules and/or who do not want or cannot access the treatment by otherestablished treatment methods (e.g., capsule, enema, endoscopy,colonoscopy, nasogastric tube, nasoenteric tube, nasal jejunal tube,etc.), and may be useful in the treatment of colitis, Crohn's,Parkinson's, Alzheimer's, post-cancer, chronic digestive issues,gastrointestinal issues (such as those associated with long Covid),autism spectrum disorder, and the like.

Manufacturing an FMT composition may include filtering a fecal sample,purifying the fecal sample by repeated separation and resuspension ofthe sediment and/or the supernatant to obtain a mixture, andfreeze-drying the mixture to obtain a freeze-dried, powderedcomposition, wherein the powdered composition may be substantiallytasteless, odorless, and colorless.

An example process includes filtering a fecal sample with a filtermedium to generate a filtrate, separating particulate matter of thefiltrate to obtain a first sediment and a first supernatant, repeatedlyresuspending the first sediment and separating particulate matter of theresuspended first sediment to obtain a second sediment and a secondsupernatant, diluting the second supernatant and repeatedly separatingparticulate matter out of the second supernatant to obtain a thirdsediment and a third supernatant, resuspending the third sediment in acryoprotectant to obtain a mixture, and freeze-drying the mixture toobtain a powdered composition, wherein the powdered composition issubstantially tasteless, odorless, and colorless.

Certain further aspects of the example process are described following,any one or more of which may be present in certain embodiments. Theexample process further includes wherein separating to obtain the firstsediment and the first supernatant is accomplished via centrifugation ata high speed. The example process further includes wherein the highspeed is greater than 1000×g. The example process further includeswherein the high speed is 8000×g. The example process further includeswherein separating to obtain the second sediment and the secondsupernatant is accomplished via centrifugation at a low speed. Theexample process further includes wherein the low speed is 1000×g. Theexample process further includes wherein separating to obtain the thirdsediment and the third supernatant is accomplished via centrifugation ata high speed. The example process further includes wherein the highspeed is greater than 1000×g. The example process further includeswherein the high speed is 8000×g. The example process further includeswherein the cryoprotectant is D-(+)-Trehalose dihydrate. The exampleprocess further includes adding a cryoprotectant to the firstsupernatant to obtain a mixture, and freeze-drying the mixture to obtaina powdered composition. The example process further includes discardingthe second sediment. The example process further includes resuspendingthe second sediment in a cryoprotectant to obtain a mixture, andfreeze-drying the mixture to obtain a powdered composition. The exampleprocess further includes discarding the third supernatant. The exampleprocess further includes adding a cryoprotectant to the thirdsupernatant to obtain a mixture, and freeze-drying the mixture to obtaina powdered composition.

An example process includes filtering a fecal sample with a filtermedium to generate a filtrate, separating particulate matter of thefiltrate to obtain a first sediment and a first supernatant, repeatedlyresuspending the first sediment and separating particulate matter of theresuspended first sediment to obtain a second sediment and a secondsupernatant, resuspending the second sediment in a cryoprotectant toobtain a mixture, and freeze-drying the mixture to obtain a powderedcomposition.

An example tasteless, odorless, and colorless powder is a product of aprocess including the steps of: filtering a fecal sample with a filtermedium to generate a filtrate, separating particulate matter of thefiltrate to obtain a first sediment and a first supernatant,resuspending the first sediment in fresh solvent and separatingparticulate matter of the resuspended first sediment to obtain a secondsediment and a second supernatant, diluting the second supernatant withfresh solvent and separating particulate matter out of the secondsupernatant to obtain a third sediment and a third supernatant,resuspending the third sediment in a cryoprotectant to obtain a mixturewhich is freeze-dried to obtain a powdered composition.

Certain further aspects of the example powder are described following,any one or more of which may be present in certain embodiments. Theexample powder further includes adding at least one of apharmaceutically-acceptable excipient, a filler, a disintegrant, apharmaceutically-acceptable carrier, a binder, or a lubricant to themixture. The example powder further includes discarding the secondsediment. The example powder further includes resuspending the secondsediment in a cryoprotectant to obtain a mixture, and freeze-drying themixture to obtain a powdered composition. The example powder furtherincludes discarding the third supernatant. The example powder furtherincludes adding a cryoprotectant to at least one of the firstsupernatant, the second supernatant, or the third supernatant to obtaina mixture, and freeze-drying the mixture to obtain a powderedcomposition. The example powder further includes filtering a fecalsample with a filter medium to generate a filtrate, separatingparticulate matter of the filtrate to obtain a first sediment and afirst supernatant, adding a cryoprotectant to the first supernatant toobtain a mixture, and freeze-drying the mixture to obtain a powderedcomposition. The example powder further includes wherein separating toobtain the first sediment and the first supernatant is accomplished viacentrifugation at a high speed. The example powder further includeswherein the high speed is greater than 1000×g. The example powderfurther includes wherein the high speed is 8000×g. The example powderfurther includes wherein the cryoprotectant is D-(+)-Trehalosedihydrate. In some embodiments, the cryoprotectant is at least one ofamino acids (e.g., alanine, glycine, proline), simple sugars (e.g.,D-(+)-Trehalose dihydrate, sucrose, glucose, lactose, ribose), dimethylsulfoxide (DMSO), or glycerol. The example powder further includessubjecting the fecal sample to a high pressure and a high temperaturesterilization.

An example tasteless, odorless, and colorless powder is a product of aprocess including the steps of: filtering a fecal sample with a filtermedium to generate a filtrate, separating particulate matter of thefiltrate to obtain a first sediment and a first supernatant, adding acryoprotectant to the first supernatant to obtain a mixture, andfreeze-drying the mixture to obtain a powdered composition. The examplepowder further includes adding at least one of apharmaceutically-acceptable excipient, a filler, a disintegrant, apharmaceutically-acceptable carrier, a binder, or a lubricant to themixture.

An example procedure including operations for treating an autismspectrum disorder (ASD) or a gastrointestinal symptom associated withASD in a subject in need thereof includes administering to the subject apowdered pharmaceutical composition including a community of fecalbacteria from a stool of a human donor, the powdered pharmaceuticalcomposition created by a process of: filtering a fecal sample with afilter medium to generate a filtrate, separating particulate matter ofthe filtrate to obtain a first sediment and a first supernatant,repeatedly resuspending the first sediment and separating particulatematter of the resuspended first sediment to obtain a second sediment anda second supernatant, diluting the second supernatant and repeatedlyseparating particulate matter out of the second supernatant to obtain athird sediment and a third supernatant, resuspending the third sedimentin a cryoprotectant to obtain a mixture, and freeze-drying the mixtureto obtain the powdered pharmaceutical composition, wherein the powderedpharmaceutical composition is substantially tasteless, odorless, andcolorless.

Certain further aspects of the example procedure are describedfollowing, any one or more of which may be present in certainembodiments. The example procedure further includes operations foradministering a maintenance dose of the powdered pharmaceuticalcomposition. The example procedure further includes operations foradministering a maintenance dose of a second powdered pharmaceuticalcomposition including a community of fecal bacteria from a stool of ahuman donor, the second powdered pharmaceutical composition created by aprocess of: adding a cryoprotectant to the first supernatant to obtain asecond mixture, and freeze-drying the second mixture to obtain a secondfreeze-dried composition that is powdered. The example procedure furtherincludes wherein administering is divided between administering aloading dose and administering a maintenance dose. The example procedurefurther includes wherein the loading dose is: a bolus dose on day one,and the bolus dose on day two. The example procedure further includeswherein the maintenance dose is a fraction of the loading dose and isadministered daily for a pre-defined number of weeks. The exampleprocedure further includes wherein the pre-defined number of weeks is16. The example procedure may further include administering a dose overa treatment period which can range from between 1 day and 4 weeks,between 1 month and 6 months, between 6 months and a year, including atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, or at least 6 months. The example procedure mayfurther include administering a dose intermittently, such as every 1month, every 4 months, every 6 months, yearly, or the like. The exampleprocedure further includes wherein the loading dose is delivered via anenema. The example procedure further includes wherein the human donor isthe subject. The example procedure further includes operations fordiscarding the second sediment. The example procedure further includesoperations for resuspending the second sediment in a cryoprotectant toobtain a mixture, and freeze-drying the mixture to obtain a powderedcomposition. The example procedure further includes operations fordiscarding the third supernatant. The example procedure further includesoperations for adding a cryoprotectant to at least one of the firstsupernatant, the second supernatant, or the third supernatant to obtaina mixture, and freeze-drying the mixture to obtain a powderedcomposition.

An example procedure including operations for treating an autismspectrum disorder (ASD) or a gastrointestinal symptom associated withASD in a subject in need thereof includes administering to the subject apowdered pharmaceutical composition including a community of fecalbacteria from a stool of a human donor, the powdered pharmaceuticalcomposition created by a process of: filtering a fecal sample with afilter medium to generate a filtrate, separating particulate matter ofthe filtrate to obtain a first sediment and a first supernatant, addinga cryoprotectant to the first supernatant to obtain a mixture, andfreeze-drying the mixture to obtain a powdered composition.

Certain further aspects of the example procedure are describedfollowing, any one or more of which may be present in certainembodiments. The example procedure further includes whereinadministering is divided between administering a loading dose andadministering a maintenance dose. The example procedure further includeswherein the loading dose is: a bolus dose on day one, and the bolus doseon day two. The example procedure further includes wherein themaintenance dose is a fraction of the loading dose and is administereddaily for a pre-defined number of weeks. The example procedure furtherincludes wherein the pre-defined number of weeks is 16. The exampleprocedure further includes wherein the loading dose is delivered via anenema. The example procedure further includes wherein the human donor isthe subject.

An example procedure including operations for treatment of a subjectwith colitis, Crohn's, Parkinson's disease, Alzheimer's disease,metabolic disorders, autism spectrum disorder, multiple sclerosis,neuropsychiatric conditions, post-cancer, chronic digestive issues,gastrointestinal issues, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), anti-aging or any condition being studied orapproved for treatment via microbial transfer includes administering tothe subject a powdered pharmaceutical composition including a communityof fecal bacteria from a stool of a human donor, the powderedpharmaceutical composition created by a process of: filtering a fecalsample with a filter medium to generate a filtrate, separatingparticulate matter of the filtrate to obtain a first sediment and afirst supernatant, repeatedly resuspending the first sediment andseparating particulate matter of the resuspended first sediment toobtain a second sediment and a second supernatant, diluting the secondsupernatant and repeatedly separating particulate matter out of thesecond supernatant to obtain a third sediment and a third supernatant,resuspending the third sediment in a cryoprotectant to obtain a mixture,and freeze-drying the mixture to obtain a the powdered pharmaceuticalcomposition, wherein the powdered pharmaceutical composition issubstantially tasteless, odorless, and colorless.

Certain further aspects of the example procedure are describedfollowing, any one or more of which may be present in certainembodiments. The example procedure further includes administering amaintenance dose of a second powdered pharmaceutical compositionincluding a community of fecal bacteria from a stool of a human donor,the second powdered pharmaceutical composition created by a process of:adding a cryoprotectant to the first supernatant to obtain a secondmixture, and freeze-drying the second mixture to obtain a secondfreeze-dried composition, wherein the second freeze-dried composition ispowdered. The example procedure further includes wherein administeringis divided between administering a loading dose and administering amaintenance dose. The example procedure further includes wherein theloading dose is: a bolus dose on day one, and the bolus dose on day two.The example procedure further includes wherein the maintenance dose is afraction of the loading dose and is administered daily for a pre-definednumber of weeks. The example procedure further includes wherein thepre-defined number of weeks is 16. The example procedure furtherincludes wherein the loading dose is delivered via an enema. The exampleprocedure further includes wherein the human donor is the subject. Theexample procedure further includes discarding the second sediment. Theexample procedure further includes resuspending the second sediment in acryoprotectant to obtain a mixture, and freeze-drying the mixture toobtain a powdered composition. The example procedure further includesdiscarding the third supernatant. The example procedure further includesadding a cryoprotectant to at least one of the first supernatant, thesecond supernatant, or the third supernatant to obtain a mixture, andfreeze-drying the mixture to obtain a powdered composition.

An example procedure including operations for treatment of a subjectwith colitis, Crohn's, Parkinson's, Alzheimer's, post-cancer, chronicdigestive issues, gastrointestinal issues, or any condition beingstudied or approved for treatment via microbial transfer includesadministering to the subject a powdered pharmaceutical compositionincluding a community of fecal bacteria from a stool of a human donor,the powdered pharmaceutical composition created by a process of:filtering a fecal sample with a filter medium to generate a filtrate,separating particulate matter of the filtrate to obtain a first sedimentand a first supernatant, adding a cryoprotectant to the firstsupernatant to obtain a mixture, and freeze-drying the mixture to obtainthe powdered pharmaceutical composition.

Certain further aspects of the example procedure are describedfollowing, any one or more of which may be present in certainembodiments. The example procedure further includes whereinadministering is divided between administering a loading dose andadministering a maintenance dose. The example procedure further includeswherein the loading dose is: a bolus dose on day one, and the bolus doseon day two. The example procedure further includes wherein themaintenance dose is a fraction of the loading dose and is administereddaily for a pre-defined number of weeks. The example procedure furtherincludes wherein the pre-defined number of weeks is 16. The exampleprocedure further includes wherein the loading dose is delivered via anenema. The example procedure further includes wherein the human donor isthe subject.

An example procedure including operations for treating an autismspectrum disorder (ASD) in a subject in need thereof includesadministering to the subject an amount of a pharmaceutical compositioneffective for treating said ASD, wherein the pharmaceutical compositionincludes a fecal microbe preparation prepared via a process including:filtering a fecal sample with a filter medium to generate a filtrate,separating particulate matter of the filtrate to obtain a first sedimentand a first supernatant, repeatedly resuspending the first sediment andseparating particulate matter of the resuspended first sediment toobtain a second sediment and a second supernatant, diluting the secondsupernatant and repeatedly separating particulate matter out of thesecond supernatant to obtain a third sediment and a third supernatant,resuspending the third sediment in a cryoprotectant to obtain a mixture,and freeze-drying the mixture to obtain a powdered composition, whereinthe powdered composition is substantially tasteless, odorless, andcolorless, wherein the subject exhibits at least a 10% reduction in ASDsymptom severity after the administering as compared to before theadministering, and based on an assessment system selected from a groupconsisting of Childhood Autism Rating Scale (CARS), Childhood AutismRating Scale 2-Standard Form (CARS2-ST), and Childhood Autism RatingScale 2-High Functioning (CARS2-HF).

Certain further aspects of the example procedure are describedfollowing, any one or more of which may be present in certainembodiments. The example procedure further includes administering amaintenance dose of a second pharmaceutical composition including acommunity of fecal bacteria from a stool of a human donor, the secondpharmaceutical composition created by the process of: adding acryoprotectant to the first supernatant to obtain a second mixture, andfreeze-drying the second mixture to obtain a second freeze-dried,powdered composition. The example procedure further includes whereinadministering is divided between administering a loading dose andadministering a maintenance dose. The example procedure further includeswherein the loading dose is: a bolus dose on day one, and the bolus doseon day two. The example procedure further includes wherein themaintenance dose is a fraction of the loading dose and is administereddaily for a pre-defined number of weeks. The example procedure furtherincludes wherein the pre-defined number of weeks is 16. The exampleprocedure further includes wherein the loading dose is delivered via anenema. The example procedure further includes wherein the fecal sampleis from the subject. The example procedure further includes discardingthe second sediment. The example procedure further includes resuspendingthe second sediment in a cryoprotectant to obtain a mixture, andfreeze-drying the mixture to obtain a powdered composition. The exampleprocedure further includes discarding the third supernatant. The exampleprocedure further includes adding a cryoprotectant to at least one ofthe first supernatant, the second supernatant, or the third supernatantto obtain a mixture, and freeze-drying the mixture to obtain a powderedcomposition.

An example procedure including operations for treating an autismspectrum disorder (ASD) in a subject in need thereof includesadministering to the subject an amount of a pharmaceutical compositioneffective for treating said ASD, wherein the pharmaceutical compositionincludes a fecal microbe preparation prepared via a process including:filtering a fecal sample with a filter medium to generate a filtrate,separating particulate matter of the filtrate to obtain a first sedimentand a first supernatant, adding a cryoprotectant to the firstsupernatant to obtain a mixture, and freeze-drying the mixture to obtaina powdered composition, wherein the subject exhibits at least a 10%reduction in ASD symptom severity after the administering as compared tobefore the administering, and based on an assessment system selectedfrom a group consisting of Childhood Autism Rating Scale (CARS),Childhood Autism Rating Scale 2-Standard Form (CARS2-ST), and ChildhoodAutism Rating Scale 2-High Functioning (CARS2-HF).

Certain further aspects of the example procedure are describedfollowing, any one or more of which may be present in certainembodiments. The example procedure further includes whereinadministering is divided between administering a loading dose andadministering a maintenance dose. The example procedure further includeswherein the loading dose is: a bolus dose on day one, and the bolus doseon day two. The example procedure further includes wherein themaintenance dose is a fraction of the loading dose and is administereddaily for a pre-defined number of weeks. The example procedure furtherincludes wherein the pre-defined number of weeks is 16. The exampleprocedure further includes wherein the loading dose is delivered via anenema. The example procedure further includes wherein the fecal sampleis from the subject.

In some aspects, the techniques described herein relate to a method ofdividing human fecal matter into useful output products, including atleast one fecal microbiota transplant (FMT) having palatable odor andflavor, the method including: a) suspending and filtering a fecal sampleacquired from a fecal donor, thereby generating a filtrate includingdesired fecal microbes of the fecal sample and a residue; b)centrifuging the filtrate at a first gravitational force of between6000×g and 10,000×g, including 8000×g, for a first centrifugation lengthof time of between 2 min and 60 min, including 20 min, and optionallyresuspending and repeating the centrifuging once, twice, or more,thereby resulting in a first supernatant and a first pellet, the firstpellet including the desired fecal microbes; c) resuspending the firstpellet and centrifuging at between 250×g and 2000×g, including 1000×g,for between 2 min and 60 min, including 20 min, and optionally decantingthe supernatant and repeating the centrifuging once, twice, or more,thereby resulting in a second supernatant and a second pellet, thesecond supernatant including the desired fecal microbes; d) centrifugingthe second supernatant or a dilution of the second supernatant atbetween 6000×g and 10,000×g, including 8000×g, for between 2 min and 60min, including 20 min, and optionally resuspending and repeating thecentrifuging once, twice, or more, thereby resulting in a thirdsupernatant and a third pellet, the third pellet including the desiredfecal microbes; e) resuspending the third pellet in the presence of acryoprotectant, thereby producing a refined suspension including thedesired fecal microbes; f) freezing the refined suspension; g)freeze-drying the frozen refined suspension, thereby providing the FMT;and h) down-stream processing the residue, the first supernatant, thesecond pellet, and/or the third supernatant, thereby producing adownstream product.

In some aspects, the techniques described herein relate to a method ofpreparing a fecal microbiota transplant (FMT) having palatable odor andflavor to facilitate non-encapsulated oral administration in largedoses, the method including the following steps: a) suspending andfiltering a fecal sample acquired from a fecal donor, thereby generatinga filtrate including desired fecal microbes of the fecal sample; b)centrifuging the filtrate at a first gravitational force of between6000×g and 10,000×g, including 8000×g, for a first centrifugation lengthof time of between 2 min and 60 min, including 20 min, and optionallyresuspending and repeating the centrifuging once, twice, or more,thereby resulting in a first supernatant and a first pellet, the firstpellet including the desired fecal microbes; c) resuspending the firstpellet and centrifuging at between 250×g and 2000×g, including 1000×g,for between 2 min and 60 min, including 20 min, and optionally decantingthe supernatant and repeating the centrifuging once, twice, or more,thereby resulting in a second supernatant and a second pellet, thesecond supernatant including the desired fecal microbes; d) centrifugingthe second supernatant or a dilution of the second supernatant atbetween 6000×g and 10,000×g, including 8000×g, for between 2 min and 60min, including 20 min, and optionally resuspending and repeating thecentrifuging once, twice, or more, thereby resulting in a thirdsupernatant and a third pellet, the third pellet including the desiredfecal microbes; e) resuspending the third pellet in the presence of acryoprotectant, thereby producing a refined suspension including thedesired fecal microbes; f) freezing the refined suspension; and g)freeze-drying the frozen refined suspension, thereby providing the FMT.

In some aspects, the techniques described herein relate to a the methodof any one of the six immediately preceding claims, wherein thetriangular test is ISO 4120.

In some aspects, the techniques described herein relate to a method oforally administering a fecal microbiota transplant (FMT) compositionwith improved compliance to a subject, the method including: orallyadministering 50 billion CFUs of the FMT composition to the subject in 2days or less.

In some aspects, the techniques described herein relate to a method oforally administering a fecal microbiota transplant (FMT) composition toa subject without requiring swallowing whole of an encapsulated dosageform, the method including: orally administering 50 billion CFUs of theFMT composition to the subject, wherein the composition is in directcontact with the subject's tongue during the orally administering.

In some aspects, the techniques described herein relate to a method oftreating a subject having been diagnosed with autism spectrum disorder(ASD), the method including: administering a predetermined loading dosefor a fecal microbiota transplant (FMT) composition under a loading doseprotocol; and administering a predetermined maintenance dose of the FMTunder a maintenance dose protocol, wherein either the loading doseprotocol or the maintenance dose protocol requires orally administering50 billion CFUs.

In some aspects, the techniques described herein relate to a method ofdividing human fecal matter into useful output products, including atleast one fecal microbiota transplant (FMT) having palatable odor andflavor, the method including: (a) suspending a fecal sample acquiredfrom a fecal donor in a first volume; (b) filtering the suspended fecalsample thereby generating a first filtrate including desired fecalmicrobes of the fecal sample and a residue; (c) filtering the suspendedfecal sample a second time thereby generating a second filtrate andadditional residue; (d) adding a second volume of fresh solvent to thesuspended fecal sample; (e) filtering the suspended fecal sample therebygenerating a third filtrate and additional residue; (f) filtering thesuspended fecal sample thereby generating a fourth filtrate andadditional residue; (g) filtering the suspended fecal sample therebygenerating a fifth filtrate and additional residue; (h) filtering thesuspended fecal sample thereby generating a sixth filtrate andadditional residue; (i) centrifuging, in separate containers, the firstfiltrate, the second filtrate, the third filtrate, the fourth filtrate,the fifth filtrate, and the sixth filtrate at a first gravitationalforce of between 6000×g and 10,000×g, including 8000×g, for a firstcentrifugation length of time of between 2 min and 60 min, including 20min and discarding a first supernatant from each of the separatecontainers; (j) adding a third volume of fresh solvent to eachcontainer, resuspending a first sediment in each container, andcentrifuging each container at the first gravitational force of between6000×g and 10,000×g, including 8000×g, for the first centrifugationlength of time of between 2 min and 60 min, including min and discardinga second supernatant from each of the separate containers; (k) adding afourth volume of fresh solvent to each container, resuspending a secondsediment in each container, and centrifuging each container at the firstgravitational force of between 6000×g and 10,000×g, including 8000×g,for the first centrifugation length of time of between 2 min and 60 min,including 20 min and discarding a third supernatant from each of theseparate containers; (l) adding a fifth volume of fresh solvent to eachcontainer, resuspending a third sediment in each container, centrifugingat a second gravitational force between 250×g and 2000×g, including1000×g, for between 2 min and min, including 20 min, decanting a fourthsupernatant from each container to a new container, and diluting thefourth supernatant in each new container with fresh solvent to reach apredetermined volume; (m) centrifuging each new container at the secondgravitational force between 250×g and 2000×g, including 1000×g, forbetween 2 min and min, including 20 min, decanting a fifth supernatantfrom each new container to a second new container, and diluting thefifth supernatant in each second new container with fresh solvent toreach the predetermined volume; (n) centrifuging each second newcontainer at the second gravitational force between 250×g and 2000×g,including 1000×g, for between 2 min and 60 min, including 20 min, anddecanting a sixth supernatant from each second new container to a thirdnew container; (o) diluting the sixth supernatant in each third newcontainer with fresh solvent to reach the predetermined volume,centrifuging each third new container at a third gravitational force ofbetween 6000×g and 10,000×g, including 8000×g, for a thirdcentrifugation length of time of between 2 min and 60 min, including 20min, discarding a seventh supernatant from each of the third newcontainers, and obtaining a fourth sediment; (p) resuspending the fourthsediment in each third new container in the presence of acryoprotectant, thereby producing a refined suspension in each third newcontainer including desired fecal microbes; f) freezing the refinedsuspension; and g) freeze-drying the frozen refined suspension, therebyproviding the FMT.

In some aspects, the techniques described herein relate to a method ofpreparing a fecal microbiota transplant (FMT) having palatable odor andflavor to facilitate non-encapsulated oral administration in largedoses, the method including the following steps: a) suspending andfiltering a fecal sample acquired from a fecal donor, thereby generatinga filtrate including desired fecal microbes of the fecal sample and aresidue; b) centrifuging the filtrate at a first gravitational force ofbetween 6000×g and 10,000×g for a first centrifugation length of time ofbetween 2 min and 60 min and optionally resuspending and repeating thecentrifuging once, twice, or more, thereby resulting in a firstsupernatant and a first pellet, the first pellet including the desiredfecal microbes; c) resuspending the first pellet and centrifuging at asecond gravitational force of between 250×g and 2000×g for a secondcentrifugation length of time of between 2 min and 60 min and optionallydecanting the supernatant and repeating the centrifuging once, twice, ormore, thereby resulting in a second supernatant and a second pellet, thesecond supernatant including the desired fecal microbes; d) centrifugingthe second supernatant or a dilution of the second supernatant at athird gravitational force of between 6000×g and 10,000×g for a thirdcentrifugation length of time of between 2 min and 60 min and optionallyresuspending and repeating the centrifuging once, twice, or more,thereby resulting in a third supernatant and a third pellet, the thirdpellet including the desired fecal microbes; e) resuspending the thirdpellet in the presence of a cryoprotectant, thereby producing a refinedsuspension including the desired fecal microbes; f) freezing the refinedsuspension; and g) freeze-drying the frozen refined suspension, therebyproviding the FMT.

In some aspects, the techniques described herein relate to a processincluding: filtering a fecal sample with a filter medium to generate afiltrate; separating particulate matter of the filtrate to obtain afirst sediment and a first supernatant; repeatedly resuspending thefirst sediment and separating particulate matter of the resuspendedfirst sediment to obtain a second sediment and a second supernatant;diluting the second supernatant and repeatedly separating particulatematter out of the second supernatant to obtain a third sediment and athird supernatant; resuspending the third sediment in a cryoprotectantto obtain a mixture; and freeze-drying the mixture to obtain a powderedcomposition, wherein the powdered composition is substantially tastelessand odorless.

In some aspects, the techniques described herein relate to a fecalmicrobiota transplant (FMT) composition for oral delivery, the FMTcomposition including an FMT including desired fecal microbes, whereinthe FMT has an odor as evaluated with a confidence of 95% to be odorlessfor more than 50% of the population using a triangular test, wherein theFMT has a flavor as evaluated with a confidence of 80% to be flavorlessfor more than 50% of the population using the triangular test.

In some aspects, the techniques described herein relate to an FMT madeby a method, the method including: a) suspending and filtering a fecalsample acquired from a fecal donor, thereby generating a filtrateincluding desired fecal microbes of the fecal sample and a residue; b)centrifuging the filtrate at a first gravitational force of between6000×g and 10,000×g for a first centrifugation length of time of between2 min and 60 min and optionally resuspending and repeating thecentrifuging once, twice, or more, thereby resulting in a firstsupernatant and a first pellet, the first pellet including the desiredfecal microbes; c) resuspending the first pellet and centrifuging at asecond gravitational force of between 250×g and 2000×g for a secondcentrifugation length of time of between 2 min and 60 min and optionallydecanting the supernatant and repeating the centrifuging once, twice, ormore, thereby resulting in a second supernatant and a second pellet, thesecond supernatant including the desired fecal microbes; d) centrifugingthe second supernatant or a dilution of the second supernatant at athird gravitational force of between 6000×g and 10,000×g for a thirdcentrifugation length of time of between 2 min and 60 min and optionallyresuspending and repeating the centrifuging once, twice, or more,thereby resulting in a third supernatant and a third pellet, the thirdpellet including the desired fecal microbes; e) resuspending the thirdpellet in the presence of a cryoprotectant, thereby producing a refinedsuspension including the desired fecal microbes; f) freezing the refinedsuspension; and g) freeze-drying the frozen refined suspension, therebyproviding the FMT.

In some aspects, the techniques described herein relate to a tastelessand odorless powder, wherein the powder is a product of a processincluding the steps of: filtering a fecal sample with a filter medium togenerate a filtrate; separating particulate matter of the filtrate toobtain a first sediment and a first supernatant; repeatedly resuspendingthe first sediment and separating particulate matter of the resuspendedfirst sediment to obtain a second sediment and a second supernatant;diluting the second supernatant and repeatedly separating particulatematter out of the second supernatant to obtain a third sediment and athird supernatant; resuspending the third sediment in a cryoprotectantto obtain a mixture; and freeze-drying the mixture to obtain a powderedcomposition.

In some aspects, the techniques described herein relate to a method oforally administering a fecal microbiota transplant (FMT) compositionwith improved compliance to a subject, the method including: orallyadministering the FMT composition to the subject, wherein the FMTcomposition includes an FMT including desired fecal microbes, whereinthe FMT has an odor as evaluated with a confidence of 95% to be odorlessfor more than 50% of the population using a triangular test, wherein theFMT has a flavor as evaluated with a confidence of 80% to be flavorlessfor more than 50% of the population using the triangular test.

In some aspects, the techniques described herein relate to a method oforally administering a fecal microbiota transplant (FMT) compositionwith improved compliance to a subject, the method including: orallyadministering 50 billion CFUs of the FMT composition to the subject in 2days or less.

In some aspects, the techniques described herein relate to a method oforally administering a fecal microbiota transplant (FMT) composition toa subject without requiring swallowing whole of an encapsulated dosageform, the method including: orally administering 50 billion CFUs of theFMT composition to the subject, wherein the composition is in directcontact with the subject's tongue during the orally administering.

These and other systems, methods, objects, features, and advantages ofthe present disclosure will be apparent to those skilled in the art fromthe following detailed description of the preferred embodiment and thedrawings.

All documents mentioned herein are hereby incorporated in their entiretyby reference. References to items in the singular should be understoodto include items in the plural, and vice versa, unless explicitly statedotherwise or clear from the text. Grammatical conjunctions are intendedto express any and all disjunctive and conjunctive combinations ofconjoined clauses, sentences, words, and the like, unless otherwisestated or clear from the context.

BRIEF DESCRIPTION OF THE FIGURES

The disclosure and the following detailed description of certainembodiments thereof may be understood by reference to the followingfigures:

FIG. 1 depicts a procedure for preparing an FMT composition.

FIG. 2 depicts a procedure for preparing an FMT composition.

FIG. 3 depicts a procedure for preparing an FMT composition.

FIG. 4 depicts a procedure for treating a disorder with an FMTcomposition.

FIG. 5 depicts a procedure for treating a disorder with an FMTcomposition.

FIG. 6 depicts a procedure for preparing an FMT composition.

FIG. 7 depicts a procedure for treating autism spectrum disorder.

FIG. 8 depicts a system for production of FMT.

FIG. 9 is a flowchart of an example method for dividing human fecalmatter into useful output products.

DETAILED DESCRIPTION

In an aspect, the disclosure herein provides compositions for FMT thatmay include fecal microbes. The term FMT is used throughout thisspecification, but it is understood that the following terms may also beused and are interchangeable: FMT is used here, but it is understoodthat the following terms or phrases are interchangeable: stooltransplantation or transfer, microbial reconstitution therapy (MRT),intestinal microbiota transplant (IMT), or microbiota transfer therapy(MTT).

As used herein, the term “fecal microbes” refers to microorganisms thatare present in the mammalian gastrointestinal system, including any ofthe gut, intestine, colon, and the like. Compositions may be prepared byprocessing fecal samples derived from mammalian stool, as describedherein. Fecal samples may comprise microbial/biological material as wellas non-living material, such as waste products, undigested foodmaterials, dead bacteria, shed host cells, proteins, carbohydrates,fats, minerals, mucus, bile, and the like.

As used herein, the term “desired fecal microbes” refers to a specificsubset of the fecal microbes that are selected based on possession ofone or more desirable properties. In some cases, the desirableproperties include desirable therapeutic properties that come fromadministration of the desired fecal microbes. In some cases, thedesirable properties include excellent survivability. In some cases, thedesired fecal microbes include microbes of one species, two species,three species, four species, five species, or six, seven, eight, nine,or ten or more species of microbe. In some cases, the desired fecalmicrobes are the full microbiome of an individual or group ofindividuals, whether selected based on overall desirability of themicrobiome or based on a particular desirability of the microbiome for aspecific purpose.

As used herein, the term “orally deliverable powder” refers to anodorless and tasteless powder form of a composition for FMT that isdesigned to be administered orally or suspended in any liquid and doesnot require encapsulation for oral delivery. In some aspects, an orallydeliverable powder is a pharmaceutical powder which is intended to beorally administered and is formulated to provide a therapeuticallyactive substance when consumed. In some aspects, an orally deliverablepowder is a pharmaceutical product, without odor or taste, which isdelivered orally without need for encapsulation, in a powdered form orsuspended in any liquid. In some aspects, orally deliverable powders areaesthetically acceptable due to reduced odor, which may be a result of adecreased presence of volatile organic molecules relative to powdersthat are not orally deliverable. Powders that are not orally deliverablemay include: any powdered FMT product that is not palatable due to odor,increased presence of volatile organic molecules, taste, or unappealingcolors; compositions for FMT that, without encapsulation, could causeharm when ingested; or a composition for FMT that without encapsulationwould cause the beneficial microbe to break down before entering the GItract and/or a location within the gut microbiome that would allow forengraftment of the fecal microbes.

In an embodiment, and referring to FIG. 1 , an example procedure 100 mayinclude an operation 102 of filtering a fecal sample with a filtermedium to generate a filtrate, an operation 104 of separatingparticulate matter of the filtrate to obtain a first sediment and afirst supernatant, an operation 108 of repeatedly resuspending the firstsediment in fresh solvent and separating particulate matter of theresuspended first sediment to obtain a second sediment and a secondsupernatant, an operation 110 of diluting the second supernatant withfresh solvent and repeatedly separating particulate matter out of thesecond supernatant to obtain a third sediment and a third supernatant,an operation 112 of resuspending the third sediment in a cryoprotectantto obtain a mixture, and an operation 114 of freeze-drying the mixtureto obtain a powdered composition, wherein the powdered composition maybe substantially tasteless, odorless, and colorless. In embodiments,fecal samples may be taken from a donor who is not the subject, or fecalsamples may be autologous, that is, taken from the donor themselves, forexample, such as from fecal matter banked prior to a time for the needfor FMT. In certain embodiments, fecal samples may be processed within atime period from collection, such as within 24 hours. Certain criteriafor the fecal sample may be applied, such as certain desired pH's or pHranges, and only certain types of stool as described on the Bristolstool chart may be processed (e.g., Type 2, Type 3, or Type 4). Aninitial visual observation for blood or mucus may also be used toexclude certain fecal samples from further processing.

A quantity of the fecal sample may be diluted with saline or otherdesired solvent in preparation for filtration. For example, 100 gm of astool sample may be diluted with 400 ml of saline or other appropriatediluent to obtain a slurry. The slurry may be filtered using anyavailable filtration technique, such as a bag filter with a perforatedor microperforated filter, membrane filters, paper filters, and thelike. In an example, the stool sample may be placed into the bag filterand the diluent added to the bag with the bag being placed into a bagmixer for further processing. The bag may be acted upon by the bag mixerfor a duration of time such as 1 minute, 2 minutes, 5 minutes, 10minutes, and the like. Processing results in a filtered solution (alsoknown as a filtrate) which may be drawn out of the bag and placed intoanother vessel for further processing. The process may be repeated forthe same duration of time, or for shorter or longer times, to obtainadditional filtered solution. Additional diluent, such as an additional100 ml, may be added to the bag and the process may be repeated for thesame duration of time, or for shorter or longer times, to obtainadditional filtered solution. In embodiments, the filtered solutionobtained in any of the processing or further processing steps may becombined for subsequent processing steps. It should be understood thatat any of the steps of dilution and/or resuspension, the amount ofdiluent utilized may be limited to improve the concentration of microbesin the sample or to optimize further processing steps, such asfreeze-drying. It should be understood that the diluent or any otherreagent used in the methods may be sterile or not.

In embodiments, the filtrate may be subjected to separation to separateparticulate matter from the filtrate and obtain a first sediment (alsoknown as settled material or a pellet) and a first supernatant. Forexample, separating to obtain the first sediment and the firstsupernatant may be accomplished via centrifugation. In an example,centrifugation may proceed at high speed, such as at a speed providing arelative centrifugal force (RCF) that is greater than 1000×g. In someembodiments, the speed is 8000×g. In other examples, as will bedescribed herein, centrifugation may proceed at lower speeds, such asspeeds below 1000×g. Without adhering to any particular thesis, it maybe understood that microbes are concentrated in the sediment whenseparated at high speed, while the metabolome, or metabolicproducts/metabolites, may be concentrated in the supernatant at lowspeeds.

In embodiments, after the initial separation that results in the firstsediment and the first supernatant, both materials may be available forfurther processing. For example, a cryoprotectant may be added to thefirst supernatant to obtain a mixture, and the mixture may befreeze-dried, such as by using a freeze dryer, to obtain a freeze-dried,powdered composition. Examples of cryoprotectants may include, but arenot limited to amino acids (e.g., alanine, glycine, proline), simplesugars (e.g., D-(+)-Trehalose dihydrate, sucrose, glucose, lactose,ribose), dimethyl sulfoxide (DMSO), glycerol, or the like. The amount ofcryoprotectant present in a composition described herein may varydepending on the cryoprotectant used and the temperature to be used forfreezing (e.g., one cryoprotectant used at −20 C may not be preferredfor use at −80 C). For example, the cryoprotectant may be 5%D-(+)Trehalose dihydrate. In an example, 32 ml of cryoprotectant may beused to resuspend or dilute the material being freeze-dried. Prior tofreeze-drying, the mixture may be subjected to a dwell time at lowtemperature, such as at −80 deg. Celsius for times ranging from 1 to 24hours or more. In embodiments, freeze-drying may occur over the courseof many hours to many days. Throughout this Specification, it should beunderstood that freeze-drying may be preceded by freezing.

In embodiments, the first sediment may be further processed byresuspending it in a fresh diluent, such as saline. For example, to thecentrifuge tube containing the pellet, fresh saline may be added and thematerial may be resuspended, such as by agitation, shaking, vibratingwith a vortex machine, or the like. For example, 45 ml of saline orother appropriate diluent may be added to the tube. Subsequent to theresuspension, the material in the tube may again be subjected toseparation, such as centrifugation at high speed, such as greater than1000×g, and at 8000×g in some embodiments. The duration of separationmay be in a range of 1 min to 25 min, 2 min to 30 min, or 30 sec to 25min. For example, the duration may be 20 min. Separation may proceed atlow temperature, such as at 4 deg. Celsius or other suitable temperaturethat preserves the biological materials being processed. Each round ofresuspension of the first sediment and centrifugation may be known as awash. After each wash, the supernatant may either be discarded orsubjected to freeze-drying in the presence of a cryoprotectant (such as32 ml of 5% D-(+)Trehalose dihydrate), as described herein. Inembodiments, any number of wash cycles may be employed. For example,once the first sediment is resuspended after the first time it iscentrifuged, no further wash cycles may be employed and the process maycontinue with steps aimed at obtaining the second sediment and secondsupernatant. In other examples, two, three, or more wash cycles may beemployed. Each wash cycle may proceed at high speed, such as at 8000×g.After the final wash cycle, the washed first sediment is resuspended,such as in a quantity of fresh saline (e.g., 45 ml), in preparation forthe next step of processing.

In embodiments, the resuspended first sediment is subject to furtherseparation, however, lower speeds may be used in this step to obtain asecond sediment and a second supernatant. The duration of separation maybe in a range of 1 min to 25 min, 2 min to 30 min, or 30 sec to 25 min.For example, the resuspended first sediment may be centrifuged at 1000×gfor 20 minutes at 4 deg. Celsius to obtain a second sediment and asecond supernatant. The second supernatant is transferred into a newtube while the second sediment is resuspended in a fresh diluent, suchas 45 ml saline, and subjected to centrifugation at low speed, such asfor a duration of 20 minutes and at a low temperature. Once again, thesupernatant is drawn off, the pellet is resuspended (e.g., in a freshsolvent/diluent), and the resuspended second sediment is once againcentrifuged. Each of the resuspension/centrifugation cycles is known asa wash cycle. After a number of wash cycles, such as between 1 and 3 or4 wash cycles, a quantity of supernatant, the washed second supernatant,that was drawn off after each cycle is available for further processing.In certain embodiments, the further processing may be to freeze-dry thewashed second supernatant in the presence of a cryoprotectant, asdescribed herein. In certain embodiments, and referring to FIG. 2 , thefurther processing may be to resuspend the second sediment in acryoprotectant to obtain a mixture 212, and freeze-dry the mixture toobtain a freeze-dried, powdered composition 214. In other embodiments,the further processing may be separation of the second supernatant toobtain a third sediment and a third supernatant. In some embodiments,the second sediment may be discarded.

In embodiments, the second supernatant may be subjected to furtherseparation. Separating to obtain the third sediment and the thirdsupernatant may be accomplished via centrifugation at a high speed, suchas speeds greater than 1000×g, such as 8000×g. The duration ofseparation may be in a range of 1 min to 25 min, 2 min to 30 min, or 30sec to 25 min. As described herein, separation may proceed for aduration of time at low temperature, such as for 20 minutes. Inembodiments, the third supernatant may be discarded, or a cryoprotectantmay be added and the third supernatant and the mixture may befreeze-dried, as described herein. In embodiments, a cryoprotectant maybe added to the third sediment and the mixture may be freeze-dried, asdescribed herein, to produce a powdered composition. The powderedcomposition may be a tasteless, odorless, and colorless powder. Thepowdered composition may be conveniently administered in a formcontaining one or more pharmaceutically acceptable carriers, such asdiluents or excipients such as fillers, binders, wetting agents,disintegrators, surface-active agents, glidants, lubricants, foodproducts, or the like. For example, given that the powdered compositionmay be substantially tasteless, odorless, and colorless, it may besprinkled on food, mixed into a smoothie/shake (e.g., such as smoothiesincluding protein powders), mixed with water, mixed intojuice/water/milk, mixed into a spread, or the like, or ingesteddirectly. In some embodiments, the powdered composition may be processedinto other orally available formulations, such as by encapsulation,tableting, and the like.

It should be understood that any of the first supernatant, the secondsupernatant, or the third supernatant may be mixed with acryoprotectant, and freeze-dried as described herein to obtain acomposition, such as a powdered composition that may be substantiallytasteless, odorless, and colorless. It should also be understood thatany of the first sediment, the second sediment, or the third sedimentmay be resuspended in a cryoprotectant, and freeze-dried as describedherein to obtain a composition, such as a powdered composition that maybe substantially tasteless, odorless, and colorless. For example, andreferring to FIG. 3 , a cryoprotectant is added to the first supernatantto obtain a mixture 302, and the mixture is freeze-dried to obtain afreeze-dried, powdered composition 304, wherein the powdered compositionmay be substantially tasteless, odorless, and colorless.

It should be understood that the fecal sample may also undergo optionalpre-processing, such as subjecting the fecal sample to a high pressureand a high temperature sterilization, such as via autoclaving.

In certain aspects, the present disclosure provides a method ofpreparing a fecal microbiota transplant (FMT). The FMT can be an orallydeliverable powder. The FMT can be tasteless. The FMT can besubstantially odorless. The FMT can have low coloration. The FMT canhave palatable odor and flavor directly in its powder form withoutrequired traditional encapsulation or masking. This FMT can facilitatenon-encapsulated oral administration in both small and large doses withconcentrated beneficial microbes. Doses of FMT may include a quantity ofdesired fecal microbes delivered orally in a given window of time.

Administering doses of FMT may be over a treatment period which canrange from between 1 day and 4 weeks, between 1 month and 6 months,between 6 months and a year, including at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, or atleast 6 months. Administering doses of FMT may include administering adose intermittently, such as every 1 month, every 4 months, every 6months, yearly, or the like. In some cases, the doses are administeredconsecutively and in other cases, doses may be staggered. In some cases,depending on the condition or health state, a small dose will beadministered over a short period including hours or days. In some cases,depending on the condition or health state, a repeated small dose willbe administered, after a large dose, over a long period including days,weeks, or years. In some cases, depending on the condition or healthstate, a repeated small dose will be administered over a long period,including days, weeks, or years. In some cases, depending on thecondition or health state, a large dose will be administered over ashort period, including hours or days. In some cases, depending on thecondition or health state, a repeated large dose will be administeredover a long period, including days, weeks, or years.

Prior to this discovery, oral administration of FMT required eitherstrong odor and flavor masking agents (e.g., chocolate milk) orencapsulation. The inventors unexpectedly discovered that carefullyprocessing fecal samples can facilitate the production of a powderedcomposition containing desired fecal microbes, which is palatable in theabsence of masking agents or physical barriers such as capsules andorally deliverable to individuals who are unable or resistant to swallowa capsule. With the understanding that this patent application may bepursued in various jurisdictions, and without wishing to be bound by anyparticular legal theory of inventiveness, it is believed that patentprotection is generally available for the discovery of a process andresulting composition for achieving all of the following: 1)transforming a typically-unpalatable material in human fecal matter intoa palatable material; 2) achieving the first goal without killingdesired fecal microbes; 3) providing a composition that can be deployedfor practical usage without requiring extreme storage conditions (e.g.,such as a liquid nitrogen-based or medical grade low temperaturefreezers); 4) achieving a degree of palatability that allows oraladministration without requiring encapsulation (or other physicalbarrier between composition and tongue); and 5) achieving a degree ofpalatability that allows non-encapsulated oral delivery withoutrequiring appreciable flavor or odor masking.

The inventors surprisingly discovered that a process using conventionalprocessing steps applied in a way that had not previously beencontemplated could overcome the shortcomings of current oral FMTdelivery systems. In an aspect, the present disclosure provides a methodof preparing a FMT having palatable odor and flavor to facilitatenon-encapsulated oral administration in large, or small, doses ofconcentrated beneficial microbes. In one particular aspect, impressivelylarge doses of microbes can be delivered. The composition for FMT issubstantially tasteless and odorless and can be easily ingested as apowder or suspended in a liquid. The method includes: a) suspending andfiltering a fecal sample acquired from a fecal donor, thereby generatinga filtrate including desired fecal microbes of the fecal sample; b)centrifuging the filtrate at a first gravitational force of between6000×g and 10,000×g, including 8000×g, for a first centrifugation lengthof time of between 2 min and 60 min, including min, and optionallyresuspending and repeating the centrifuging once, twice, or more,thereby resulting in a first supernatant and a first pellet, the firstpellet including the desired fecal microbes; c) resuspending the firstpellet and centrifuging at a second gravitational force of between 250×gand 2000×g, including 1000×g, for a second centrifugation length of timeof between 2 min and 60 min, including 20 min, and optionally decantingthe supernatant and repeating the centrifuging once, twice, or more,thereby resulting in a second supernatant and a second pellet, thesecond supernatant including the desired fecal microbes; d) centrifugingthe second supernatant or a dilution of the second supernatant at athird gravitational force of between 6000×g and 10,000×g, including8000×g, for a third centrifugation length of time of between 2 min and60 min, including 20 min, and optionally resuspending and repeating thecentrifuging once, twice, or more, thereby resulting in a thirdsupernatant and a third pellet, the third pellet including the desiredfecal microbes; e) resuspending the third pellet in the presence of acryoprotectant, thereby producing a refined suspension including thedesired fecal microbes; f) freezing the refined suspension; and g)freeze-drying the frozen refined suspension, thereby providing the FMT.In some specific instances, the method can further optionally include:h) downstream processing the residue, the first supernatant, the secondpellet, and/or the third supernatant, thereby producing a downstreamproduct.

The fecal sample itself can be harvested from a single source or avariety of subjects and/or sources. The fecal sample can be harvestedfrom a single region or a variety of regions.

In some cases, the fecal sample is harvested from an individual that isdifferent than the subject to which the FMT will be administered.

The fecal sample can be mixed with other fecal samples at any stagethroughout the method or divided into sub-samples, whether before orafter suspending and filtering, or the various filtrates, supernatants,pellets, and the like can be combined or divided to facilitateprocessing efficiency or quality.

In some cases, the fecal sample can be harvested from a donor that has adesirable diagnosis history with respect to one or more conditions. Insome cases, the donor has a diagnosis history that shows a lack ofautism spectrum disorder (ASD). In some cases, the donor is screened asnot having ASD. In some cases, the donor is diagnosed as cognitivelynormal or cognitively above average.

In some cases, the fecal sample can be harvested from a donor on thebasis of meeting a suitability threshold as determined from screeningdata, including medical background and health history. Some factors usedto include or exclude fecal sample donors may include one or more of:obesity, a presence of beneficial fecal flora, childbirth type (e.g.,vaginal delivery, C-section, etc.), having certain diseases orconditions (e.g., chronic infectious disease(s), known exposure to HIVor viral hepatitis, autoimmune disease(s), gastrointestinal disease orsymptoms, atopic disease(s), metabolic disorders, mood disorders andneuro-atypical, neurodegenerative disorder(s), chronic pain syndrome,malignancies, etc.), whether or not breastfed, activity/fitness level,donor age, diversity of diet, type of diet (e.g., omnivorous, carnivore,vegan, vegetarian, etc.), amount of lifetime use of certain agents(e.g., antibiotics, antifungals, antivirals, immune-suppressants,chemotherapy, etc.), allergy history, tobacco/cannabis smoking historyincluding secondhand history, international travel, ADHD/ADD treatment,sleep issues (e.g. insomnia, frequent waking, nightmares), country ofresidence, various parameters related to blood and stool (e.g., presenceof ova/parasites, viruses (including Multidrug-Resistant Organisms(MDROs): ESBL, MRSA, VRE), fungi (e.g., C. difficile toxin B by PCR),and/or active infections (e.g., enteric pathogens)), or other serologictest results (e.g., Complete Blood Count (CBC) & Comprehensive MetabolicPanel (CMP), HIV (type 1 and 2), Hepatitis A virus IgM, Hepatitis Bsurface antigen, Hepatitis C virus antibody, Treponema pallidum (Cascadewith reflex to RPR)).

In some cases, the fecal sample is held in quarantine for a period oftime (e.g., 30 days, 60 days, 90 days, 120 days, or the like) to ensureall blood and stool screening passes any set standards or criteria.

In some cases, the fecal sample is included or excluded on the basis ofa criteria. For example, the fecal sample one criteria may be its Typeas based on a Bristol stool chart or other classification scale. Inanother example, the criteria may be exhibition of a selected pH or pHranges of the fecal sample. In another example, the criteria may be thepresence or absence of blood or mucus. It should be understood that thestep of applying criteria for inclusion or exclusion may precede any ofthe processing steps described herein.

Suspending the fecal sample can be performed according to techniquesunderstood in the art, including but not limited to aggressive mixing,vortexing, bag mixing, and the like. Without wishing to be bound by anyparticular theory, it is believed that skilled artisans will recognizethat suspending a fecal sample broadly relates to techniques forreducing particle size and making fecal matter into a liquid compositionhaving suspended fecal particulates. In some cases, suspension proceedsuntil a homogenous solution is achieved.

Filtering a fecal sample can be performed according to techniquesunderstood in the art, including but not limited to, conventionalfiltration, suction filtration, bag filtration, and the like. Withoutwishing to be bound by any particular theory, it is believed thatskilled artisans will recognize that filtering a fecal sample broadlyrelates to techniques for removing larger particulate matter from afecal sample.

The filtering can be performed with a particle size cutoff that allowsthe desired fecal microbes to be adequately separated from larger piecesof fecal matter, as would be appreciated by a skilled artisan. Ingeneral, the filtering has a particle size cutoff of 150 microns, 200microns, 250 microns, 300 microns, 350 microns, or 400 microns. In somecases, the filtering has a particle size cutoff of 400 microns. In somecases, the filtering has a particle size cutoff of 250 microns. In somecases, the filtering has a particle size cutoff of 150 microns. In somecases, the filtering involves filtering products out that are largerthan 250 microns and keeping everything sized under 250 microns. In somecases, the filtering involves filtering products out that are largerthan 150 microns. In some cases, the filtering involves filteringproducts out that are larger than 400 microns. Filtering may proceedwith a limit on heat and oxygen exposure for the material to be filteredand filtered material. For example, filtering may proceed at atemperature between 1° C. and 10° C. In some cases, filtering mayproceed at a temperature between 3° C. and 5° C. filtering may proceedat a temperature of 4° C.

The various centrifugation steps are performed under generallyconventional conditions using generally conventional instrumentation.The inventor surprisingly discovered that the sequencing and control ofdifferent centrifugation steps can provide exceptional performancequality. It is understood that any of the centrifugation steps describedherein may be performed on any volume of filtrate, such as volumessuitable for the available centrifugation instrumentation.

Following the suspending and filtering, the filtrate is centrifuged(also referred to herein as a first centrifugation or the centrifugationof step b)) at a first gravitational force for a first centrifugationlength of time. Centrifuging the filtrate results in a first pellet anda first supernatant. The desired fecal microbes are contained in thefirst pellet. The centrifuging can optionally include one, two, three,four, five, or more resuspensions with fresh saline (or other suspendingliquid) and decantings of supernatants.

To be clear and for the avoidance of doubt, the present disclosurecovers: i) situations where the first centrifugation involves only asingle centrifugation step that results in a first supernatant; ii)situations where the first centrifugation involves a first instance ofcentrifugation, after which the supernatant is gathered, the pellet isresuspended, and a second instance of centrifugation is performed withthe same and/or similar operational parameters—the supernatant from thefirst instance can be combined with the supernatant from the secondinstance to define the “first supernatant” as a whole or the firstsupernatant may be only the supernatant from the first instance ofcentrifugation; iii) situations where the first centrifugation involvesa first instance of centrifugation, after which the supernatant isgathered, the pellet is resuspended, and multiple subsequent instancesof centrifugation, decanting, and resuspension are performed with thesame and/or similar operational parameters—the supernatant from thefirst instance can be combined with the multiple supernatants from themultiple subsequent instances to define the “first supernatant” as awhole or the first supernatant may be only the supernatant from thefirst instance of centrifugation; iv) slight variations incentrifugation parameters (e.g. plus or minus 10%), while still beingconsidered part of the “same” first centrifugation.

In some cases, the first centrifugation includes two resuspensions anddecantings. In some cases, a total first centrifugation time of thefirst centrifugation of step b) can be between 6 min and 180 min,including 60 min.

The specific operational parameters of the first centrifugation step canbe tailored for desired performance, including for the selection and/orexclusion of certain constituents. This disclosure expresslycontemplates an active selection step in the methods disclosed herein,where the particular parameters of the first centrifugation are selectedto provide a specific desired result in one or more of the outputcompositions.

In general, the first gravitational force is between 6000×g and10,000×g, between 7000×g and 9000×g, or between 7500×g and 8500×g,including 8000×g. In general, the first centrifugation length of time isbetween 2 min and 60 min, between 5 min and 50 min, between 10 min and45 min, between 15 min and 30 min, or between 15 min and 25 min,including 20 min.

In certain cases, the first gravitational force is between 6000×g and10,000×g, including 8000×g. In certain cases, the first gravitationalforce is between 7000×g and 9000×g, including 8000×g. In certain cases,the first gravitational force is between 7500×g and 8500×g, including8000×g. In certain cases, the first length of time is between 2 min and60 min, including 20 min. In certain cases, the first length of time isbetween 10 min and 30 min, including 20 min. In certain cases, the firstlength of time is between 15 min and 25 min, including 20 min. Incertain cases, the first gravitational force is between 6000×g and10,000×g, including 8000×g, and the first length of time is between 2min and 60 min, including 20 min. In certain cases, the firstgravitational force is between 7000×g and 9000×g, including 8000×g, andthe first length of time is between 10 min and 30 min, including 20 min.In certain cases, the first gravitational force is between 7500×g and8500×g, including 8000×g, and the first length of time is between 15 minand 25 min, including 20 min.

Following the first centrifugation of step b), the first pellet isresuspended and centrifuged (also referred to herein as a secondcentrifugation or the centrifugation of step c)) at a secondgravitational force for a second centrifugation length of time. Theresult is a second pellet and a second supernatant. The desired fecalmicrobes are contained in the second supernatant.

As discussed above with the first centrifugation of step b), the secondcentrifugation of step c) can be repeated multiple times, but with aslight modification due to the second supernatant being retained (alongwith the desired fecal microbes) rather than the first pellet. It shouldbe appreciated that the centrifugation can be repeated zero, one, two,three, four, five, or more times, gathering and/or discarding the pelleteach time and optionally adding additional liquid to maintain originalvolume. In some cases, the centrifugation is repeated twice. In somecases, a total second centrifugation time of the second centrifugationof step c) can be between 6 min and 180 min, including 60 min.

The specific operational parameters of the second centrifugation stepcan be tailored for desired performance, including for the selectionand/or exclusion of certain constituents. This disclosure expresslycontemplates an active selection step in the methods disclosed herein,where the particular parameters of the second centrifugation areselected to provide a specific desired result in one or more of theoutput compositions.

In general, the second gravitational force is between 250×g and 2000×g,between 500×g and 1500×g, or between 750×g and 1250×g, including 1000×g.In general, the second centrifugation length of time is between 2 minand 60 min, between 5 min and min, between 10 min and 45 min, between 15min and 30 min, or between 15 min and min, including 20 min.

In certain cases, the second gravitational force is between 250×g and2000×g, including 1000×g. In certain cases, the second gravitationalforce is between 500×g and 1500×g, including 1000×g. In certain cases,the second gravitational force is between 750×g and 1250×g, including1000×g. In certain cases, the second length of time is between 2 min and60 min, including 20 min. In certain cases, the second length of time isbetween 10 min and 30 min, including 20 min. In certain cases, thesecond length of time is between 15 min and 25 min, including 20 min. Incertain cases, the second gravitational force is between 250×g and2000×g, including 1000×g, and the second length of time is between 2 minand 60 min, including 20 min. In certain cases, the second gravitationalforce is between 500×g and 1500×g, including 1000×g, and the secondlength of time is between 10 min and 30 min, including 20 min. Incertain cases, the second gravitational force is between 750×g and1250×g, including 1000×g, and the second length of time is between 15min and 25 min, including 20 min.

Following the second centrifugation of step c), the second supernatantor a dilution of the second supernatant is centrifuged (also referred toherein as a third centrifugation or the centrifugation of step d)) at athird gravitational force for a third centrifugation length of time. Theresult is a third pellet and a third supernatant. The desired fecalmicrobes are contained in the third pellet.

As discussed above with the first centrifugation of step b), the thirdcentrifugation of step d) can be repeated multiple times, but with aslight modification due to the third supernatant being retained (alongwith the desired fecal microbes) rather than the first pellet. It shouldbe appreciated that the centrifugation can be repeated zero, one, two,three, four, five, or more times, gathering and/or discarding the pelleteach time and optionally adding additional liquid to maintain originalvolume. In some cases, the third centrifugation is repeated twice. Insome cases, the third centrifugation is not repeated in step d). In somecases, a total third centrifugation time of the third centrifugation ofstep d) can be between 2 min and 180 min, including 20 min.

The specific operational parameters of the third centrifugation step canbe tailored for desired performance, including for the selection and/orexclusion of certain constituents. This disclosure expresslycontemplates an active selection step in the methods disclosed herein,where the particular parameters of the third centrifugation are selectedto provide a specific desired result in one or more of the outputcompositions.

In general, the third gravitational force is between 6000×g and10,000×g, between 7000×g and 9000×g, or between 7500×g and 8500×g,including 8000×g. In general, the third centrifugation length of time isbetween 2 min and 60 min, between 5 min and 50 min, between 10 min and45 min, between 15 min and 30 min, or between 15 min and 25 min,including 20 min.

In certain cases, the third gravitational force is between 6000×g and10,000×g, including 8000×g. In certain cases, the third gravitationalforce is between 7000×g and 9000×g, including 8000×g. In certain cases,the third gravitational force is between 7500×g and 8500×g, including8000×g. In certain cases, the third length of time is between 2 min and60 min, including 20 min. In certain cases, the third length of time isbetween 10 min and 30 min, including 20 min. In certain cases, the thirdlength of time is between 15 min and 25 min, including 20 min. Incertain cases, the third gravitational force is between 6000×g and10,000×g, including 8000×g, and the third length of time is between 2min and 60 min, including 20 min. In certain cases, the thirdgravitational force is between 7000×g and 9000×g, including 8000×g, andthe third length of time is between 10 min and 30 min, including 20 min.In certain cases, the third gravitational force is between 7500×g and8500×g, including 8000×g, and the third length of time is between 15 minand 25 min, including 20 min.

Following the third centrifugation of step d), the third pellet isresuspended in the presence of a cryoprotectant, thereby producing arefined suspension. The refined suspension includes the desired fecalmicrobes.

The cryoprotectant can be one or more of the cryoprotectants articulatedabove. In some cases, the cryoprotectant is D-(+)Trehalose dihydrate.The cryoptectant can be added in an amount that results in the FMTcontaining between 0.1% and 20% by weight of the cryoprotectant,including 5% by weight.

The cryoprotectant can be added at effectively any point in the method,so long as the subsequent processing steps would not lead to itsremoval. While the cryoprotectant may most conventionally be addedduring step e), it is contemplated for the cryoprotectant to be added atother times. The cryoprotectant can be added to the first pellet priorto or during step c) or to the resuspended first pellet during step c);the cryoprotectant can be added to the second supernatant prior to orduring step d) or to the optionally diluted second supernatant duringstep d); and/or the cryoprotectant can be added to the third pelletprior to or during step e) or to the resuspended third pellet duringstep e). In some cases, the cryoprotectant is added prior to step e). Insome cases, the cryoprotectant is added during step e).

Following the resuspension, the refined suspension is frozen.

Following the freezing of the refined suspension, the frozen refinedsuspension is freeze-dried, resulting in the FMT. Freeze-drying may takeplace over a number of days, such as at least 6 days, at least 5 days,at least 4 days, at least 2 days, or at least 1 day.

The inventors surprisingly discovered that the method retains a highpercentage of the desired fecal microbes from the original fecal sample.In other words, the inventors unexpectedly discovered that the methoddisclosed herein has a high survival rate for the desired fecalmicrobes, given that the degree to which the odor and flavor have beeneliminated is most typically associated with harsh treatments thatrequire the killing of all or most living things, including desirablefecal microbes. In some cases, at least 95% of the desired fecalmicrobes that are present in the fecal matter is retained in the FMTfollowing the method. In some cases, at least 75% of the desired fecalmicrobes that are present in the fecal matter is retained in the FMTfollowing the method. In some cases, at least 50% of the desired fecalmicrobes that are present in the fecal matter is retained in the FMTfollowing the method. In some cases, the count of desired fecal microbespresent in the fecal matter or FMT is determined by flow cytometry.

The inventors were able to achieve an unexpectedly strong probioticcount in the FMT. Unless the context clearly dictates otherwise, alltotal probiotic counts described herein refer to a probiotic count ofdesired fecal microbes. In some cases, the FMT has a total probioticcount of at least 50 billion colony forming units (CFUs) following themethod. In some cases, the FMT has a total probiotic count of at least10 billion CFUs following the method. In some cases, the FMT has a totalprobiotic count of at least 1 billion CFUs following the method. In somecases, the probiotic count of desired fecal microbes is determined byflow cytometry.

One of the two truly landmark achievements of the present disclosure isthat the FMT produced by the methods described herein is odorless to asignificant portion of the population. As measured by a triangularcomparison test, such as the one described in ISO 4120 (ISO 4120:2021,Sensory analysis—Methodology—Triangle test), which is herebyincorporated by reference in its entirety, the FMT has an odor asevaluated with a confidence of 95% to be odorless for more than 70% ofthe population. In some cases, the FMT has an odor as evaluated with aconfidence of 95% to be odorless for more than 60% of the population. Insome cases, the FMT has an odor as evaluated with a confidence of 95% tobe odorless for more than 50% of the population.

The other of the truly landmark achievements of the present disclosureis that the FMT produced by the methods described herein is tasteless toa significant portion of the population. As measured by a triangularcomparison test, such as the one described in ISO 4120, the FMT has aflavor as evaluated with a confidence of 80% to be flavorless for morethan 70% of the population. In some cases, the FMT has a flavor asevaluated with a confidence of 80% to be flavorless for more than 60% ofthe population. In some cases, the FMT has a flavor as evaluated with aconfidence of 80% to be flavorless for more than 50% of the population.

Without wishing to be bound by any particular theory, and despite havingtest results that indicate some degree of retention of color, the FMT isa very low color powder and/or produces a very low color liquid whendissolved.

In some cases, the compositions described herein can be process andhandled in one environment, sealed within that environment, transportedto a different environment for centrifugation, and then either returnedto the first environment or taken to a third, different environment.This approach enables the use of a clean environment for processing thesamples and a non-sterile environment for the centrifugation.

The FMT is stable when stored under a variety of conditions, as measuredby one or more of a retention of a desired activity level, a viability,an engraftment, or an efficacy of the gut microbiome over time duringstorage. In some aspects, the FMT may be considered shelf-stable. Ingeneral, storing the FMT under predetermined storage conditions for apredetermined length of time retains the desired activity level.

The predetermined storage conditions can be −80° C. to 4° C. Thepredetermined storage conditions can be −20° C. to 0° C. Thepredetermined storage conditions can be 0° C. to 4° C. The predeterminedstorage conditions can be room temperature. It should be understood thatroom temperature describes a temperature of an ambient environment, suchas a temperature between about 68° F. to 75° F., including 70° F. Thepredetermined storage time can be more than 2 years, including more than5 years. The predetermined storage time can be 2 years. Thepredetermined storage time can be 1 year. The predetermined storage timecan be between 3 months and 5 years. The predetermined storage time canbe 6 months. The predetermined storage time can be 3 months. The desiredactivity level can be 100%. The desired activity level can be 85%. Thedesired activity level can be 70%.

The method can optionally further include: h) downstream processing theresidue, the first supernatant, the second pellet, and/or the thirdsupernatant, thereby producing a downstream product.

Downstream processing the residue can include autoclaving the residue.

Downstream processing the first supernatant can include concentratingthe first supernatant, dehydrating the first supernatant, freeze-dryingthe first supernatant, or a combination thereof. The decantedsupernatant(s) from the first centrifugation of step b) is the firstsupernatant and optionally includes the supernatants from resuspensionsin the instances where they occur. The decanted supernatants can be keptseparate or combined.

Downstream processing the second pellet can include autoclaving and/orsuspending the second pellet.

Downstream processing the third supernatant includes concentrating thethird supernatant, dehydrating the third supernatant, freeze-drying thethird supernatant, or a combination thereof.

Given that the desired fecal microbes are the entity that is beingtransplanted, and given that the residue, the first supernatant, thesecond pellet, and the third supernatant substantially lack the desiredfecal microbes, they all could conventionally be viewed as wastestreams, but the inventors surprisingly discovered that the residue, thefirst supernatant, the second pellet, and/or the third supernatant couldbe further processed for useful purposes, thereby enhancing the overallefficiency of the process.

The method disclosed herein can be fully automated, as would beappreciated by an industrial automation engineer.

The steps of extracting desirable microbes may be performed in a shortduration of time. For example, the overall process of extractingmicrobes may be performed in less than four hours, less than threehours, less than two hours, or less than 90 minutes.

In an aspect, the present disclosure provides a therapeutic compositionfor oral administration to a subject. The therapeutic composition caninclude the FMT or the powder described herein. In addition, thetherapeutic composition can include a pharmaceutically acceptablecarrier. Though one of the principal advantages of the presentcomposition is the ability to be used without additional additives, thepresence of such additives are still expressly contemplated within thescope of the invention. One example of a suitable additive for use withthe compositions described herein is an enteric coating that is tailoredto enhance survivability of the desirable fecal microbes as theynavigate the subject's GI tract.

Products produced by any of the processes described herein may be usefulin the treatment of a number of disorders, such as autism spectrumdisorder (ASD), a gastrointestinal symptom associated with ASD, colitis,Crohn's, Parkinson's, Alzheimer's, post-cancer, chronic digestiveissues, gastrointestinal issues, or any condition being studied orapproved for treatment via microbial transfer. In an embodiment, andreferring to FIG. 4 , an example procedure 400 for treating an autismspectrum disorder (ASD) or a gastrointestinal symptom associated withASD in a subject in need thereof may include an operation 402 ofadministering to the subject a powdered pharmaceutical composition, suchas an amount of a pharmaceutical composition effective for treating saidASD, comprising a community of fecal bacteria from a stool of a humandonor, the powdered pharmaceutical composition created by a process ofthe subsequent steps of the procedure 400, including an operation 404 offiltering a fecal sample with a filter medium to generate a filtrate, anoperation 408 of separating particulate matter of the filtrate to obtaina first sediment and a first supernatant, an operation 410 of repeatedlyresuspending the first sediment and separating particulate matter of theresuspended first sediment to obtain a second sediment and a secondsupernatant, an operation 412 of diluting the second supernatant andrepeatedly separating particulate matter out of the second supernatantto obtain a third sediment and a third supernatant, an operation 414 ofresuspending the third sediment in a cryoprotectant to obtain a mixture,and an operation 418 of freeze-drying the mixture to obtain afreeze-dried composition that is powdered, wherein the powderedcomposition may be substantially tasteless, odorless, and colorless. Theprocedure 400 may include administering a loading dose initially,followed by maintenance doses, or extended treatment, over a timeperiod. In embodiments, the loading dose and the maintenance dose may beprepared via the same procedures. In embodiments, and as previouslydescribed herein, any powdered compositions derived from freeze-dryingany of the first supernatant, the second supernatant, the thirdsupernatant, the first sediment, the second sediment, or the thirdsediment may be useful in the procedure and may be administered as theloading dose and/or the maintenance dose. Similarly, any of the firstsupernatant, the second supernatant, the third supernatant, the firstsediment, the second sediment, or the third sediment may be discarded.

For example, and referring to FIG. 5 , a procedure 500 for treating anautism spectrum disorder (ASD) or a gastrointestinal symptom associatedwith ASD in a subject in need thereof may include an operation 402 ofadministering to the subject a powdered pharmaceutical compositioncomprising a community of fecal bacteria from a stool of a human donor,the powdered pharmaceutical composition created by subsequent steps ofthe procedure 500, including an operation 404 of filtering a fecalsample with a filter medium to generate a filtrate, an operation 408 ofseparating particulate matter of the filtrate to obtain a first sedimentand a first supernatant, an operation 502 of adding a cryoprotectant tothe first supernatant to obtain a mixture, and an operation 504 offreeze-drying the mixture to obtain a freeze-dried, powdered, whereinthe powdered composition may be substantially tasteless, odorless, andcolorless.

In an embodiment, the loading dose may be a bolus dose on day one, and abolus dose on day two. In the example, the bolus doses may be the sameor different. In an example of an encapsulated powdered composition, thebolus dose may be at least 10 capsules, at least 20 capsules, at least30 capsules, or at least 35 capsules. In an example of anon-encapsulated product, the bolus dose may be at least 10 vials, atleast 20 vials, at least 30 vials, or at least 35 vials. The maintenancedose may be a fraction of the bolus dose and may be administered dailyfor a pre-defined number of weeks, such as four weeks, eight weeks,sixteen weeks, or the like, or for other durations such as months (e.g.,6 months, 9 months, etc.) or years (e.g., 1 year, 2 years, 5 years,etc.). In some aspects, the maintenance dose schedule may be a periodicone. In some cases, after a set amount of time on a prophylactic basisor to address reoccurring symptoms, the maintenance dose may bere-initiated. For example, a 4 week treatment may be delivered every 6months.

In some embodiments, the loading dose may be delivered via an enema. Insome embodiments, the human donor is the subject. It should beunderstood that the dose and/or duration of dosing of the powderedcomposition may be variable or customized based on any number offactors, such as size, weight, age, severity of disease or symptoms,tolerance levels, diet, ability to swallow, prior treatment, additionaltreatments/supplements, comorbidities, or the like. For example, a dosemay vary in accordance with a current or desired rating on at least oneof the Childhood Autism Rating Scale (CARS), the Childhood Autism RatingScale 2-Standard Form (CARS2-ST), or the Childhood Autism Rating Scale2-High Functioning (CARS2-HF). In another example, the powderedcomposition may be complemented by a specific diet and/or concomitantpharmacological treatment, and the dose or duration of dosing may varydepending on aspects of the diet and/or concomitant pharmacologicaltreatment.

In embodiments, treating subjects with an autism spectrum disorder (ASD)or a gastrointestinal symptom associated with ASD using the powderedcompositions described herein may result in the subject exhibiting areduction in ASD symptom severity after the administering as compared tobefore the administering. For example, an assessment may be administeredto the subject prior to administering treatment, such as an assessmentsystem selected from a group consisting of Childhood Autism Rating Scale(CARS), Childhood Autism Rating Scale 2-Standard Form (CARS2-ST),Childhood Autism Rating Scale 2-High Functioning (CARS2-HF), AutismTreatment Evaluation Checklist (ATEC)), Gastrointestinal symptoms(Gastrointestinal Symptom Rating Scale (GSRS)), Bristol scale, qualityof life (Quality of Life in Autism Questionnaire (QoLA)), or anotherknown or not-yet-known assessment. After a duration of treatmentcomprising administering one or more doses of the powdered composition,the selected assessment may be re-administered to determine if there hasbeen an improvement in any of the administered assessments or areduction in severity of observed symptoms. For example, re-assessmentmay take place at 30 days post-treatment, 60 days post-treatment, 90days post-treatment, 120 days post-treatment, or 180 dayspost-treatment. Additionally, a number of subjective measures may beassessed, such as parents' view on change, change inmedication/supplements/diet/overall health/stool, or the like. Forexample, an amount of a pharmaceutical composition effective fortreating an ASD may be an amount that results in a 10% reduction inseverity of ASD symptoms, such as determined by the selected assessmentsystem. In embodiments, the effective amount may be delivered in aloading dose, or may be delivered in a combination of a loading and oneor more maintenance doses.

In embodiments, a long-term maintenance dose may also be administeredafter completing the loading dose and/or maintenance doses. In someembodiments, the loading and maintenance doses together may beconsidered FMT, while the long-term maintenance may be consideredpost-FMT maintenance. Post-FMT maintenance may be dosed hourly, daily,weekly, monthly, or the like. Post-FMT maintenance doses may be derivedfrom any of the processes described herein. Post-FMT maintenance dosesmay be based on metabolome products, such as supernatant-derivedcompositions, or compositions derived from autoclaved fecal samples, forexample.

In embodiments, either of the procedure 400 or procedure 500, andrelated process steps described herein, as well as any of the powderedcompositions described herein, may also be useful in the treatment of asubject with C. difficile infection, colitis, Crohn's, cancer,post-cancer, chronic digestive issues, gastrointestinal issues (such asthose associated with long Covid or other viral disease), inflammatorybowel disease (IBD), inflammatory bowel syndrome (IBS), metabolicdisorders (i.e. type 2 diabetes, insulin resistance, metabolicsyndrome), Alzheimer's Disease, Parkinson's disease, age-related changes(i.e. cognition, frailty, mood), anti-aging multiple sclerosisneuropsychiatric conditions (i.e. anxiety, depression), or any conditionbeing studied or approved for treatment via microbial transfer. Itshould be understood that the dose of the powdered composition may bevariable or customized based on any number of factors, such as size,weight, age, disease or disorder being treated, severity of disease orsymptoms, tolerance levels, ability to swallow, prior treatment, or thelike.

Referring now to FIG. 6 , a method 600 of dividing human fecal matterinto useful output products, including at least one fecal microbiotatransplant (FMT) having palatable odor and flavor may include a step 602of suspending and filtering a fecal sample acquired from a fecal donor,thereby generating a filtrate including desired fecal microbes of thefecal sample and a residue. The method 600 may further include a step604 of centrifuging the filtrate at a first gravitational force ofbetween 6000×g and including 8000×g, for a first centrifugation lengthof time of between 2 min and 60 min, including 20 min, and optionallyresuspending and repeating the centrifuging once, twice, or more,thereby resulting in a first supernatant and a first pellet, the firstpellet including the desired fecal microbes. The method may furtherinclude a step 608 of resuspending the first pellet and centrifuging ata second gravitational force of between 250×g and 2000×g, including1000×g, for a second centrifugation length of time of between 2 min and60 min, including 20 min, and optionally decanting the supernatant andrepeating the centrifuging once, twice, or more, thereby resulting in asecond supernatant and a second pellet, the second supernatant includingthe desired fecal microbes. The method may further include a step 610 ofcentrifuging the second supernatant or a dilution of the secondsupernatant at a third gravitational force of between 6000×g and10,000×g, including 8000×g, for a third centrifugation length of time ofbetween 2 min and 60 min, including 20 min, and optionally resuspendingand repeating the centrifuging once, twice, or more, thereby resultingin a third supernatant and a third pellet, the third pellet includingthe desired fecal microbes. The method may further include a step 612 ofresuspending the third pellet in the presence of a cryoprotectant,thereby producing a refined suspension including the desired fecalmicrobes. The method may further include a step 614 of freezing therefined suspension and a step of 618 of freeze-drying the frozen refinedsuspension, thereby providing a fecal microbiota transplant. The method600 may optionally include a step 620 of down-stream processing theresidue, the first supernatant, the second pellet, and/or the thirdsupernatant, thereby producing a downstream product.

Referring now to FIG. 7 , a method 700 of treating a subject having beendiagnosed with autism spectrum disorder (ASD) may include administeringa predetermined loading dose for a fecal microbiota transplant (FMT)composition under a loading dose protocol 702, and administering apredetermined maintenance dose of the FMT under a maintenance doseprotocol 704. In certain embodiments, either the loading dose protocolor the maintenance dose protocol requires orally administering 50billion CFUs.

Referring now to FIG. 8 , an embodiment of a system 800 for producingFMT having palatable odor and flavor is depicted. The system 800 mayinclude a filtration system 802, such as a BagMixer, into which acontainer 804, such as a bag, is placed to mix a suspended fecal sampleand filter the suspension to obtain a filtrate, which is placed into acontainer 810 for subsequent processing. The filtrate is separated, suchas in a centrifuge 808, to obtain supernatant and sediment/pellet,either of which may be useful for further downstream processing. Inembodiments, where the sediment/pellet is used, it may be resuspended infresh solvent, such as by use of a vortex machine, shaker, or the like.In embodiments, if the supernatant is used, it may be decanted andoptionally diluted for further processing. Products obtained after anyof the steps of filtration, dilution, centrifugation, mixing, decanting,and/or resuspension may be mixed with a cryoprotectant, frozen, such asin a freezer 812, and freeze-dried, such as in a freeze dryer 814,thereby providing the FMT.

FIG. 9 is a flowchart of an example method 900 for dividing human fecalmatter into useful output products, including at least one fecalmicrobiota transplant (FMT) having palatable odor and flavor. At step902, the method 900 involves suspending a fecal sample acquired from afecal donor in a first volume. At step 904, the method 900 involvesfiltering the suspended fecal sample thereby generating a filtrateincluding desired fecal microbes of the fecal sample. At step 908, themethod 900 involves centrifuging the filtrate at a first gravitationalforce of 8000×g for a first centrifugation length of time of 20 min,retaining a first sediment, and discarding a first supernatant. At step910, the method 900 involves adding fresh solvent, resuspending thefirst sediment, and centrifuging each container at the firstgravitational force for the first centrifugation length of time,retaining a second sediment, and discarding a second supernatant. Atstep 912, the method 900 involves adding fresh solvent, resuspending thesecond sediment, and centrifuging the first gravitational force for thefirst centrifugation length of time, retaining a third sediment, anddiscarding a third supernatant from each of the separate containers. Atstep 914, the method 900 involves adding fresh solvent, resuspending thethird sediment, and centrifuging at a second gravitational force of1000×g for a second centrifugation length of time of 20 min, retaining afourth supernatant, and discarding a fourth sediment. At step 918, themethod 900 involves centrifuging the fourth supernatant or a dilutionthereof at the second gravitational force for the second centrifugationlength of time, retaining a fifth supernatant, and discarding a fifthsediment. At step 920, the method 900 involves centrifuging the fifthsupernatant or a dilution thereof at the second gravitational force forthe second centrifugation length of time, retaining a sixth supernatant,and discarding a sixth sediment. At step 922, the method 900 involvescentrifuging the sixth supernatant or a dilution thereof at the firstgravitational force for the first centrifugation length of time,discarding a seventh supernatant, and obtaining a seventh sediment. Atstep 924, the method 900 involves resuspending the seventh sediment inthe presence of a cryoprotectant, thereby producing a refinedsuspension. At step 928, the method 900 involves freezing the refinedsuspension. At step 930, the method 900 involves and freeze-drying thefrozen refined suspension, thereby providing the FMT.

It should be understood that powdered compositions derived from anysupernatant (e.g., first, second, or third), or any sediment (e.g.,first, second, or third) may be substantially tasteless, odorless, andcolorless. In some embodiments, powdered compositions derived from anysupernatant (e.g., first, second, or third), or any sediment (e.g.,first, second, or third) may not be substantially tasteless, odorless,and colorless. In some embodiments, powdered compositions derived fromany fecal samples that have been autoclaved may not be substantiallytasteless, odorless, and colorless. For example, a first supernatantthat is freeze-dried into a powder may not be substantially tasteless,odorless, and colorless and may be encapsulated.

The methods and systems described herein may transform physical and/oror intangible items from one state to another. The methods and systemsdescribed herein may also transform data representing physical and/orintangible items from one state to another.

The elements described and depicted herein, including in flow charts,block diagrams, and/or operational descriptions, depict and/or describespecific example arrangements of elements for purposes of illustration.However, the depicted and/or described elements, the functions thereof,and/or arrangements of these, may be implemented on machines, such asthrough computer executable transitory and/or non-transitory mediahaving a processor capable of executing program instructions storedthereon, and/or as logical circuits or hardware arrangements. Examplearrangements of programming instructions include at least: monolithicstructure of instructions; standalone modules of instructions forelements or portions thereof; and/or as modules of instructions thatemploy external routines, code, services, and so forth; and/or anycombination of these, and all such implementations are contemplated tobe within the scope of embodiments of the present disclosure Examples ofsuch machines include, without limitation, personal digital assistants,laptops, personal computers, mobile phones, other handheld computingdevices, medical equipment, wired or wireless communication devices,transducers, chips, calculators, satellites, tablet PCs, electronicbooks, gadgets, electronic devices, devices having artificialintelligence, computing devices, networking equipment, servers, routersand the like. Furthermore, the elements described and/or depictedherein, and/or any other logical components, may be implemented on amachine capable of executing program instructions. Thus, while theforegoing flow charts, block diagrams, and/or operational descriptionsset forth functional aspects of the disclosed systems, any arrangementof program instructions implementing these functional aspects arecontemplated herein. Similarly, it will be appreciated that the varioussteps identified and described above may be varied, and that the orderof steps may be adapted to particular applications of the techniquesdisclosed herein. Additionally, any steps or operations may be dividedand/or combined in any manner providing similar functionality to thedescribed operations. All such variations and modifications arecontemplated in the present disclosure. The methods and/or processesdescribed above, and steps thereof, may be implemented in hardware,program code, instructions, and/or programs or any combination ofhardware and methods, program code, instructions, and/or programssuitable for a particular application. Example hardware includes adedicated computing device or specific computing device, a particularaspect or component of a specific computing device, and/or anarrangement of hardware components and/or logical circuits to performone or more of the operations of a method and/or system. The processesmay be implemented in one or more microprocessors, microcontrollers,embedded microcontrollers, programmable digital signal processors orother programmable device, along with internal and/or external memory.The processes may also, or instead, be embodied in an applicationspecific integrated circuit, a programmable gate array, programmablearray logic, or any other device or combination of devices that may beconfigured to process electronic signals. It will further be appreciatedthat one or more of the processes may be realized as a computerexecutable code capable of being executed on a machine readable medium.

The computer executable code may be created using a structuredprogramming language such as C, an object oriented programming languagesuch as C++, or any other high-level or low-level programming language(including assembly languages, hardware description languages, anddatabase programming languages and technologies) that may be stored,compiled or interpreted to run on one of the above devices, as well asheterogeneous combinations of processors, processor architectures, orcombinations of different hardware and computer readable instructions,or any other machine capable of executing program instructions.

Thus, in one aspect, each method described above and combinationsthereof may be embodied in computer executable code that, when executingon one or more computing devices, performs the steps thereof. In anotheraspect, the methods may be embodied in systems that perform the stepsthereof, and may be distributed across devices in a number of ways, orall of the functionality may be integrated into a dedicated, standalonedevice or other hardware. In another aspect, the means for performingthe steps associated with the processes described above may include anyof the hardware and/or computer readable instructions described above.All such permutations and combinations are contemplated in embodimentsof the present disclosure.

EXAMPLES

Some embodiments described herein are further illustrated by thefollowing example which should not be construed as limiting,

Example 1. Preparation of an FMT Composition

To prepare an FMT composition, stool samples were evaluated for at leastpH, stool type (e.g., stool type as identified from a Bristol chart),presence of a contaminant (e.g., blood, mucus, other), and color.Suitable samples, as determined by one or more of the intendedcondition(s) to treat, the intended subject(s) to treat, a regulatorypolicy, a practitioner's instruction, or the like, were furtherprocessed. 100 grams of stool sample was added to a BagPage filter bag,400 ml of saline was added, and then the bag was placed in a BagMixerfor 2 minutes. 50 ml of the filtered solution was withdrawn andtransferred to a tube. The BagMixer was run again for 2 minutes andanother 50 ml of filtered solution was withdrawn and placed in a tube.100 ml of saline was added to the BagPage filter bag, the BagMixer wasrun again twice for 2 minutes each time, and 50 ml of filter solutionwas withdrawn after each time and placed in a tube. Another 100 ml ofsaline was added to the BagPage filter bag, the BagMixer was run againtwice for 2 minutes each time, and 50 ml of filter solution waswithdrawn after each time and placed in a tube. All six tubes with 50 mleach of filtered solution were placed in a centrifuge and run at 8000×gfor 30 minutes at 4° C. The supernatant was discarded and each tube wasfilled to the 45 ml line with fresh saline and the settled material wasresuspended using the vortex machine. Centrifugation, supernatantdiscarding, and resuspension were repeated twice.

After the third resuspension, the tubes were once again subjected tocentrifugation at 1000×g for 20 min at 4° C. The supernatant wastransferred to a clean tube and the volume was adjusted with a salinesolution up to 45 ml. Centrifugation, supernatant transfer, and volumeadjustment to 45 ml was repeated twice. After the third volumeadjustment, the tubes were once again subjected to centrifugation at8000×g for 20 min at 4° C. The supernatant was discarded. 32 ml of 5%D-(+)-Trehalose dihydrate was added to each tube and vortexed until thesettled material was resuspended.

1.5 ml of the resuspended material was transferred into 1.5 ml vials andfrozen at −80° C. overnight. The vial weights ranged between 60 and 140mg. The frozen vials were removed from the freezer and freeze-dried forabout three days with opened lids. The vials were sealed once complete.

The finished powder product was assessed for odor, flavor, and colorusing ISO 4120 standards, among other evaluative techniques, by anindependent contracted third party.

In one batch of tests, the powder was diluted in 5 ounces of water andsubjected to a triangular test. The triangular test operates bycomparing two different samples, A and B. Each panelist in the test isgiven three samples, either two A samples and one B sample or one Asample and two B samples. Panelists are asked to identify which sampleis different from the other two. The number of correct answers werecounted and statistically analyzed under ISO 4120 standards to assessthe degree to which individuals can taste, smell, or see a differencebetween the inventive composition and a control (tap water).

For the odor assessment, 11 of 30 panelists correctly identified thedifferent sample. According to the ISO 4120 table of triangular testoutcomes, we conclude with a confidence of 95% that the composition isodorless for more than 70% of the population.

For the taste assessment, 12 of 30 panelists correctly identified thedifferent sample. According to the ISO 4120 table of triangular testoutcomes, we conclude with a confidence of 80% that the product istasteless for more than 70% of the population.

For the color assessment, 27 of 30 panelists correctly identified thedifferent sample. While this is a subpar result in terms of showingcolorlessness when compared with a control, human perception of color ishighly skewed by the presence of a local comparison. We understand thisintuitively based on a few common observations. As one example, opticalillusions can be created by changing the surrounding color environment,so two identical colors and shades appear very different, despite beingidentical, due to environmental context. As another example, teethwhitening advertisements observe that the true lack of whiteness ofteeth cannot be observed without comparison to a truer white, such as atissue—this is quite literally the concept that we believe we are seeinghere, where the control sample is the “white tissue”, and where the testsample is the individual's teeth. If a panelist were to view the testsample in a vacuum without the comparison (e.g., to look in the mirrorwithout a tissue for whiteness comparison), they may conclude that thecomposition is colorless. However, if they view the test sample next toa comparison (e.g., to use the tissue), they may show the results thatwe achieved.

While the disclosure has been disclosed in connection with the preferredembodiments shown and described in detail, various modifications andimprovements thereon will become readily apparent to those skilled inthe art. Accordingly, the spirit and scope of the present disclosure isnot to be limited by the foregoing examples, but is to be understood inthe broadest sense allowable by law.

1.-221. (canceled)
 222. A method of orally administering a fecalmicrobiota transplant (FMT) composition with improved compliance to asubject, the method comprising: orally administering the FMT compositionto the subject, wherein the FMT composition comprises an FMT includingdesired fecal microbes, wherein the FMT has an odor as evaluated with aconfidence of 95% to be odorless for more than 50% of the populationusing a triangular test, wherein the FMT has a flavor as evaluated witha confidence of 80% to be flavorless for more than 50% of the populationusing the triangular test.
 223. The method of claim 222, wherein the FMTcomposition is not encapsulated.
 224. The method of claim 222, whereinthe FMT composition is in direct contact with the subject's tongueduring the orally administering.
 225. The method of claim 222, wherein50 billion CFUs are administered in 2 days or less.
 226. The method ofclaim 222, wherein orally administering comprises one or more of thefollowing: i) orally administering a comestible having mixed therein apowder form of the composition; ii) orally administering a comestiblehaving applied thereto a powder form of the composition; iii) orallyadministering a solubilized form of the composition; or a combinationthereof.
 227. The method of claim
 226. wherein orally administeringcomprises orally administering a comestible having mixed therein apowder form of the composition.
 228. The method of claim 226, whereinorally administering comprises orally administering a comestible havingapplied thereto a powder form of the composition.
 229. The method ofclaim 226, wherein orally administering comprises orally administering asolubilized form of the composition.
 230. The method of claim 222,wherein orally administering comprises administering a loading dose andone or more maintenance doses.
 231. The method of claim 222, wherein theFMT has a color that is different than human fecal matter.
 232. Themethod of claim 222, wherein the FMT has an odor as evaluated with aconfidence of 95% to be odorless for more than 70% of the populationusing a triangular test.
 233. The method of claim 222, wherein the FMThas a flavor as evaluated with a confidence of 80% to be flavorless formore than 50% of the population using the triangular test.
 234. Themethod of claim 222, wherein the subject has a condition for whichmicrobial transfer is a known treatment.
 235. The method of claim 222,wherein the FMT composition is not co-administered with a flavor or odormasking agent.
 236. The method of claim 222, wherein the FMT is apowder.
 237. The method of claim 222, wherein the FMT is freeze-dried.238. The method of claim 222, wherein the triangular test isInternational Organization for Standardization (ISO)
 4120. 239. Themethod of claim 222, wherein the FMT is made by a method, the methodcomprising: a) suspending and filtering a fecal sample acquired from afecal donor, thereby generating a filtrate including desired fecalmicrobes of the fecal sample and a residue; b) centrifuging the filtrateat a first gravitational force of between 6000×g and 10,000×g for afirst centrifugation length of time of between 2 min and 60 min andoptionally resuspending and repeating the centrifuging once, twice, ormore, thereby resulting in a first supernatant and a first pellet, thefirst pellet including the desired fecal microbes; c) resuspending thefirst pellet and centrifuging at a second gravitational force of between250×g and 2000×g for a second centrifugation length of time of between 2min and 60 min and optionally decanting the supernatant and repeatingthe centrifuging once, twice, or more, thereby resulting in a secondsupernatant and a second pellet, the second supernatant including thedesired fecal microbes; d) centrifuging the second supernatant or adilution of the second supernatant at a third gravitational force ofbetween 6000×g and 10,000×g for a third centrifugation length of time ofbetween 2 min and 60 min and optionally resuspending and repeating thecentrifuging once, twice, or more, thereby resulting in a thirdsupernatant and a third pellet, the third pellet including the desiredfecal microbes; e) resuspending the third pellet in the presence of acryoprotectant, thereby producing a refined suspension including thedesired fecal microbes; f) freezing the refined suspension; and g)freeze-drying the frozen refined suspension, thereby providing the FMT.240. A method of orally administering a fecal microbiota transplant(FMT) composition with improved compliance to a subject, the methodcomprising: orally administering 50 billion CFUs of the FMT compositionto the subject in 2 days or less.
 241. A method of orally administeringa fecal microbiota transplant (FMT) composition to a subject withoutrequiring swallowing whole of an encapsulated dosage form, the methodcomprising: orally administering 50 billion CFUs of the FMT compositionto the subject, wherein the composition is in direct contact with thesubject's tongue during the orally administering.